Salmonella infect their animal hosts by entering into and traversing the intestinal epithelial barrier. Penetration of the epithelium is primarily achieved by direct invasion of host epithelial cells using a panel of effector molecules secreted through a Type III secretion apparatus. SipC, which forms part of the tip of this apparatus, has long cytoplasmic N- and C-termini, which nucleate the assembly of actin filament networks that are required for bacterial engulfment. Using a yeast two-hybrid screen, we identified a number of host proteins that interact selectively with the C-terminus of SipC. These include proteins that regulate actin assembly and dynamics, as well as proteins that mediate vesicular transport. We hypothesize that Salmonella uses SipC to nucleate the assembly of signaling and cytoskeletal machinery at sites of bacterial attachment to coordinate actin remodeling with the delivery of new membrane to form the nascent phagosome. This hypothesis will be explored in Specific Aim 1. Salmonella infection triggers the recruitment of inflammatory monocytes to the intestine, which have an important role in clearance of the infection. However, Salmonella can also use phagocytes as vehicles for their systemic dissemination, and the success or failure of the host response lies in the balance between these two processes. We are examining the role of Focal Adhesion Kinase (FAK) in the migration and function of monocytic cells. Using mice that conditionally lack FAK in cells of the myeloid lineage, we found that infiltration of inflammatory macrophages into the Peyer's patches and mesenteric lymph nodes is impaired in the absence of FAK, and that this surprisingly correlates with reduced bacterial colonization of all tissues examined. Conversely, neutrophil infiltration into the same tissues is enhanced, suggesting a differential regulation of macrophage and neutrophil recruitment to infected tissues. We hypothesize that inflammatory macrophages are necessary for bacterial survival in the tissue microenvironment, by providing a protective niche for bacterial replication and dissemination, and that in the absence of this niche bacteria are more efficiently killed by infiltrating neutrophils. This hypothesis will be tested in Specific Aim 2. Recent evidence indicates the existence of a novel population of lysozyme-expressing dendritic cells unique to the Peyer's patch, that are the first cells targeted by Salmonella upon transiting the intestinal epithelium. However nothing is known about the role of these cells in Salmonella infection. Characterization of these cells forms the basis for Specific Aim 3. Together, the results of these studies will provide significant new insight into the mechanisms used by Salmonella to infect their animal hosts, and the role of the innate immune response in combating the infection. The molecular details that emerge from these studies may provide new targets for next generation antibiotics or therapies for the prevention of Salmonellosis. PUBLIC HEALTH RELEVANCE: Infection with pathogenic Salmonella strains causes symptoms ranging from gastritis to potentially fatal systemic disease such as Typhoid Fever. The goals of this research are to define the host cell machinery that is subverted by Salmonella to enter the intestinal epithelium and spread systemically, and to characterize the innate immune responses to Salmonella infection.